Plants and seeds of high oil corn variety HOI002

ABSTRACT

According to the invention, there is provided seed and plants of the corn variety designated HOI002. This invention thus relates to the plants, seeds and tissue cultures of the variety HOI002, and to methods for producing a corn plant produced by crossing a corn plant of variety HOI002 with itself or with another corn plant, such as a plant of another variety. This invention further relates to corn seeds and plants produced by crossing plants of variety HOI002 with plants of another variety, such as another inbred line, and to crosses with related species. This invention further relates to the inbred and hybrid genetic complements of plants of variety HOI002, and also to the SSR and isozyme typing profiles of corn variety HOI002.

[0001] This application claims the priority of U.S. Provisional PatentApplication Ser. No. 60/343,964, filed Dec. 28, 2001, the entiredisclosure of which is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the field of cornbreeding. In particular, the invention relates to inbred corn seed andplants of the variety designated HOI002, and tissue cultures thereof, aswell as methods of use thereof.

[0004] 2. Description of Related Art

[0005] The goal of field crop breeding is to combine various desirabletraits in a single variety/hybrid. Such desirable traits include greateryield, better stalks, better roots, resistance to insecticides,herbicides, pests, and disease, tolerance to heat and drought, reducedtime to crop maturity, better agronomic quality, higher nutritionalvalue, and uniformity in germination times, stand establishment, growthrate, maturity, and fruit size.

[0006] Breeding techniques take advantage of a plant's method ofpollination. There are two general methods of pollination: a plantself-pollinates if pollen from one flower is transferred to the same oranother flower of the same plant. A plant cross-pollinates if pollencomes to it from a flower on a different plant.

[0007] Corn plants (Zea mays L.) can be bred by both self-pollinationand cross-pollination. Both types of pollination involve the cornplant's flowers. Corn has separate male and female flowers on the sameplant, located on the tassel and the ear, respectively. Naturalpollination occurs in corn when wind blows pollen from the tassels tothe silks that protrude from the tops of the ear shoot.

[0008] Plants that have been self-pollinated and selected for type overmany generations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

[0009] The development of uniform corn plant hybrids requires thedevelopment of homozygous inbred plants, the crossing of these inbredplants, and the evaluation of the crosses. Pedigree breeding andrecurrent selection are examples of breeding methods used to developinbred plants from breeding populations. Those breeding methods combinethe genetic backgrounds from two or more inbred plants or various otherbroad-based sources into breeding pools from which new inbred plants aredeveloped by selfing and selection of desired phenotypes. The newinbreds are crossed with other inbred plants and the hybrids from thesecrosses are evaluated to determine which of those have commercialpotential.

[0010] The pedigree breeding method involves crossing two genotypes.Each genotype can have one or more desirable characteristics lacking inthe other; or, each genotype can complement the other. If the twooriginal parental genotypes do not provide all of the desiredcharacteristics, other genotypes can be included in the breedingpopulation. Superior plants that are the products of these crosses areselfed and selected in successive generations. Each succeedinggeneration becomes more homogeneous as a result of self-pollination andselection. Typically, this method of breeding involves five or moregenerations of selfing and selection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc.After at least five generations, the inbred plant is consideredgenetically pure.

[0011] Backcrossing can also be used to improve an inbred plant.Backcrossing transfers a specific desirable trait from one inbred ornon-inbred source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred (A)(recurrent parent) to a donor inbred (non-recurrent parent), whichcarries the appropriate locus or loci for the trait in question. Theprogeny of this cross are then mated back to the superior recurrentparent (A) followed by selection in the resultant progeny for thedesired trait to be transferred from the non-recurrent parent. Afterfive or more backcross generations with selection for the desired trait,the progeny are heterozygous for loci controlling the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

[0012] A single cross hybrid corn variety is the cross of two inbredplants, each of which has a genotype that complements the genotype ofthe other. The hybrid progeny of the first generation is designated F₁.Typically, F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, is manifested in many polygenic traits,including markedly improved yields, better stalks, better roots, betteruniformity and better insect and disease resistance. In the developmentof hybrids only the F₁ hybrid plants are typically sought. An F₁ singlecross hybrid is produced when two inbred plants are crossed. A doublecross hybrid is produced from four inbred plants crossed in pairs (A×Band C×D) and then the two F₁ hybrids are crossed again (A×B)×(C×D).

[0013] The development of a hybrid corn variety involves three steps:(1) the selection of plants from various germplasm pools; (2) theselfing of the selected plants for several generations to produce aseries of inbred plants, which, although different from each other, eachbreed true and are highly uniform; and (3) crossing the selected inbredplants with unrelated inbred plants to produce the hybrid progeny (F₁).During the inbreeding process in corn, the vigor of the plantsdecreases. Vigor is restored when two unrelated inbred plants arecrossed to produce the hybrid progeny (F₁). An important consequence ofthe homozygosity and homogeneity of the inbred plants is that the hybridbetween any two inbreds is always the same. Once the inbreds that give asuperior hybrid have been identified, hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained. Conversely, much of the hybrid vigor exhibited by F₁ hybridsis lost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock. It is not generally beneficialfor farmers to save seed of F₁ hybrids. Rather, farmers purchase F₁hybrid seed for planting every year.

[0014] North American farmers plant tens of millions of acres of corn atthe present time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides a corn plant of thevariety designated HOI002. Also provided are corn plants having all thephysiological and morphological characteristics of the inbred cornvariety HOI002. The inbred corn plant of the invention may furthercomprise, or have, a cytoplasmic or nuclear factor that is capable ofconferring male sterility or otherwise preventing self-pollination, suchas by self-incompatibility. Parts of the corn plant of the presentinvention are also provided, for example, pollen obtained from an inbredplant and an ovule of the inbred plant.

[0016] The invention also concerns seed of the inbred corn varietyHOI002. A sample of this seed has been deposited under ATCC AccessionNo. PTA-3788. The inbred corn seed of the invention may be provided asan essentially homogeneous population of inbred corn seed of the varietydesignated HOI002. Essentially homogeneous populations of inbred seedare those that consist essentially of the particular inbred seed, andare generally free from substantial numbers of other seed, so that theinbred seed forms between about 90% and about 100% of the total seed,and preferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 99.9% of inbredseed, as measured by seed grow outs. This corresponds to currentcommercial practice among the leading companies in the seed industry.

[0017] Therefore, in the practice of the present invention, inbred seedgenerally forms at least about 97% of the total seed. However, even if apopulation of inbred corn seed was found, for some reason, to containabout 50%, or even about 20% or 15% of inbred seed, this would still bedistinguished from the small fraction (generally less than 2% andpreferably less than 1%) of inbred seed that may be found within apopulation of hybrid seed, e.g., within a commercial bag of hybrid seed.In such a bag of hybrid seed offered for sale, Federal regulationsrequire that the hybrid seed be at least about 95% of the total seed, orbe labeled as a mixture. In the most preferred practice of theinvention, the female inbred seed that may be found within a bag ofhybrid seed will be about 1% of the total seed, or less, and the maleinbred seed that may be found within a bag of hybrid seed will benegligible, i.e., will be on the order of about a maximum of 1 per100,000, and usually less than this value.

[0018] The population of inbred corn seed of the invention can furtherbe particularly defined as being essentially free from hybrid seed. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of inbred corn plants designated HOI002.

[0019] In another aspect of the invention, single locus converted plantsof variety HOI002 are provided. The single transferred locus maypreferably be a dominant or recessive allele. Preferably, the singletransferred locus will confer such traits as male sterility, yieldstability, waxy starch, yield enhancement, industrial usage, herbicideresistance, insect resistance, resistance to bacterial, fungal, nematodeor viral disease, male fertility, and enhanced nutritional quality. Thesingle locus may be a naturally occurring maize gene introduced into thegenome of the variety by backcrossing, a natural or induced mutation, ora transgene introduced through genetic transformation techniques. Whenintroduced through transformation, a single locus may comprise one ormore transgenes integrated at a single chromosomal location.

[0020] In yet another aspect of the invention, an inbred corn plant ofthe variety designated HOI002 is provided, wherein acytoplasmically-inherited trait has been introduced into said inbredplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continue to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

[0021] In another aspect of the invention, a tissue culture ofregenerable cells of a plant of variety HOI002 is provided. The tissueculture will preferably be capable of regenerating plants capable ofexpressing all of the physiological and morphological characteristics ofthe variety, and of regenerating plants having substantially the samegenotype as other plants of the variety. Examples of some of thephysiological and morphological characteristics of the variety HOI002include characteristics related to yield, maturity, and kernel quality,each of which is specifically disclosed herein. The regenerable cells insuch tissue cultures will preferably be derived from embryos,meristematic cells, immature tassels, microspores, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, orstalks, or from callus or protoplasts derived from those tissues. Stillfurther, the present invention provides corn plants regenerated from thetissue cultures of the invention, the plants having all thephysiological and morphological characteristics of variety HOI002.

[0022] In yet another aspect of the invention, processes are providedfor producing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is aplant of the variety designated HOI002. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second corn plant ofa different, distinct variety to provide a hybrid that has, as one ofits parents, the inbred corn plant variety HOI002. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

[0023] In a preferred embodiment of the invention, the first step in“crossing” comprises planting, preferably in pollinating proximity,seeds of a first and second parent corn plant, and preferably, seeds ofa first inbred corn plant and a second, distinct inbred corn plant.Where the plants are not in pollinating proximity, pollination cannevertheless be accomplished by transferring a pollen or tassel bag fromone plant to the other as described below.

[0024] A second step comprises cultivating or growing the seeds of saidfirst and second parent corn plants into plants that bear flowers. Cornbears both male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant.

[0025] A third step comprises preventing self-pollination of the plants,i.e., preventing the silks of a plant from being fertilized by any plantof the same variety, including the same plant. This is preferably doneby emasculating the male flowers of the first or second parent cornplant, (i.e., treating or manipulating the flowers so as to preventpollen production, in order to produce an emasculated parent cornplant), Self-incompatibility systems are also used in some hybrid cropsfor the same purpose. Self-incompatible plants still shed viable pollenand can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same variety.

[0026] A fourth step comprises allowing cross-pollination to occurbetween the first and second parent corn plants. When the plants are notin pollinating proximity, this is done by placing a bag, usually paperor glassine, over the tassels of the first plant and another bag overthe silks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

[0027] The present invention also provides corn seed and plants producedby a process that comprises crossing a first parent corn plant with asecond parent corn plant, wherein at least one of the first or secondparent corn plants is a plant of the variety designated HOI002. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation (F₁) hybrid corn seed and plants producedby crossing an inbred in accordance with the invention with another,distinct inbred. The present invention further contemplates seed of anF₁ hybrid corn plant. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid corn plant and seed thereof.

[0028] In still yet another aspect of the invention, the geneticcomplement of the corn plant variety designated HOI002 is provided. Thephrase “genetic complement” is used to refer to the aggregate ofnucleotide sequences, the expression of which sequences defines thephenotype of, in the present case, a corn plant, or a cell or tissue ofthat plant. A genetic complement thus represents the genetic make up ofan inbred cell, tissue or plant, and a hybrid genetic complementrepresents the genetic make up of a hybrid cell, tissue or plant. Theinvention thus provides corn plant cells that have a genetic complementin accordance with the inbred corn plant cells disclosed herein, andplants, seeds and diploid plants containing such cells.

[0029] In still yet another aspect, the present invention provideshybrid genetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

[0030] In still yet another aspect, the present invention provides amethod of producing an inbred corn plant derived from the corn varietyHOI002, the method comprising the steps of: (a) preparing a progenyplant derived from corn variety HOI002, wherein said preparing comprisescrossing a plant of the corn variety HOI002 with a second corn plant,and wherein a sample of the seed of corn variety HOI002 has beendeposited under ATCC Accession No. PTA-3788; (b) crossing the progenyplant with itself or a second plant to produce a seed of a progeny plantof a subsequent generation; (c) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant; and (d) repeating steps (c) and (d) for an additional2-10 generations, including at least about 3, 5, 7, 9, 10 or moregenerations, to produce an inbred corn plant derived from the cornvariety HOI002. In the method, it may be desirable to select particularplants resulting from step (c) for continued crossing according to steps(b) and (c). By selecting plants having one or more desirable traits, aninbred corn plant derived from the corn variety HOI002 is obtained thatpossesses some of the desirable traits of corn variety HOI002 as wellpotentially other selected traits.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS OF PLANTCHARACTERISTICS

[0031] Barren Plants: Plants that are barren, i.e., lack an ear withgrain, or have an ear with only a few scattered kernels.

[0032] Cg: Colletotrichum graminicola rating. Rating times 10 isapproximately equal to percent total plant infection.

[0033] CLN: Corn Lethal Necrosis (combination of Maize Chlorotic MottleVirus and Maize Dwarf Mosaic virus) rating: numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible.

[0034] Cn: Corynebacterium nebraskense rating. Rating times 10 isapproximately equal to percent total plant infection.

[0035] Cz: Cercospora zeae-maydis rating. Rating times 10 isapproximately equal to percent total plant infection.

[0036] Dgg: Diatraea grandiosella girdling rating (values are percentplants girdled and stalk lodged).

[0037] Dropped Ears: Ears that have fallen from the plant to the ground.

[0038] Dsp: Diabrotica species root ratings (1=least affected to9=severe pruning).

[0039] Ear-Attitude: The attitude or position of the ear at harvestscored as upright, horizontal, or pendant.

[0040] Ear-Cob Color: The color of the cob, scored as white, pink, red,or brown.

[0041] Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

[0042] Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

[0043] Ear-Diameter: The average diameter of the ear at its midpoint.

[0044] Ear-Dry Husk Color: The color of the husks at harvest scored asbuff, red, or purple.

[0045] Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

[0046] Ear-Husk Bract: The length of an average husk leaf scored asshort, medium, or long.

[0047] Ear-Husk Cover: The average distance from the tip of the ear tothe tip of the husks. Minimum value no less than zero.

[0048] Ear-Husk Opening: An evaluation of husk tightness at harvestscored as tight, intermediate, or open.

[0049] Ear-Length: The average length of the ear.

[0050] Ear-Number Per Stalk: The average number of ears per plant.

[0051] Ear-Shank Internodes: The average number of internodes on the earshank.

[0052] Ear-Shank Length: The average length of the ear shank.

[0053] Ear-Shelling Percent: The average of the shelled grain weightdivided by the sum of the shelled grain weight and cob weight for asingle ear.

[0054] Ear-Silk Color: The color of the silk observed 2 to 3 days aftersilk emergence scored as green-yellow, yellow, pink, red, or purple.

[0055] Ear-Taper (Shape): The taper or shape of the ear scored asconical, semi-conical, or cylindrical.

[0056] Ear-Weight: The average weight of an ear.

[0057] Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

[0058] ER: Ear rot rating (values approximate percent ear rotted).

[0059] Final Stand Count: The number of plants just prior to harvest.

[0060] GDUs: Growing degree units are calculated herein by the BargerMethod, where the heat units for a 24-h period are calculated as GDUs=[(Maximum daily temperature+Minimum daily temperature)/2]-50. Thehighest maximum daily temperature used is 86° F. and the lowest minimumtemperature used is 50° F.

[0061] GDUs to Shed: The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50% ofthe plants shedding pollen as measured from time of planting. GDUs toshed is determined by summing the individual GDU daily values fromplanting date to the date of 50% pollen shed.

[0062] GDUs to Silk: The number of growing degree units for an inbredline or hybrid to have approximately 50% of the plants with silkemergence as measured from time of planting. GDUs to silk is determinedby summing the individual GDU daily values from planting date to thedate of 50% silking.

[0063] Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0064] Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0065] Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0066] Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0067] Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

[0068] HtG: Chlorotic-lesion type resistance. +=indicates the presenceof Ht chlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−=indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

[0069] Kernel-Aleurone Color: The color of the aleurone scored as white,pink, tan, brown, bronze, red, purple, pale purple, colorless, orvariegated.

[0070] Kernel-Cap Color: The color of the kernel cap observed at drystage, scored as white, lemon-yellow, yellow, or orange.

[0071] Kernel-Endosperm Color: The color of the endosperm scored aswhite, pale yellow, or yellow.

[0072] Kernel-Endosperm Type: The type of endosperm scored as normal,waxy, or opaque.

[0073] Kernel-Grade: The percent of kernels that are classified asrounds.

[0074] Kernel-Length: The average distance from the cap of the kernel tothe pedicel.

[0075] Kernel-Number Per Row: The average number of kernels in a singlerow.

[0076] Kernel-Pericarp Color: The color of the pericarp scored ascolorless, red-white crown, tan, bronze, brown, light red, cherry red,or variegated.

[0077] Kernel-Row Direction: The direction of the kernel rows on the earscored as straight, slightly curved, spiral, or indistinct (scattered).

[0078] Kernel-Row Number: The average number of rows of kernels on asingle ear.

[0079] Kernel-Side Color: The color of the kernel side observed at thedry stage, scored as white, pale yellow, yellow, orange, red, or brown.

[0080] Kernel-Thickness: The distance across the narrow side of thekernel.

[0081] Kernel-Type: The type of kernel scored as dent, flint, orintermediate.

[0082] Kernel-Weight: The average weight of a predetermined number ofkernels.

[0083] Kernel-Width: The distance across the flat side of the kernel.

[0084] Kz: Kabatiella zeae rating. Rating times 10 is approximatelyequal to percent total plant infection.

[0085] Leaf-Angle: Angle of the upper leaves to the stalk scored asupright (0 to 30 degrees), intermediate (30 to 60 degrees), or lax (60to 90 degrees).

[0086] Leaf-Color: The color of the leaves 1 to 2 weeks afterpollination scored as light green, medium green, dark green, or verydark green.

[0087] Leaf-Length: The average length of the primary ear leaf.

[0088] Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

[0089] Leaf-Marginal Waves: A rating of the waviness of the leaf margin1 to 2 weeks after pollination. Rated as none, few, or many.

[0090] Leaf-Number: The average number of leaves of a mature plant.Counting begins with the cotyledonary leaf and ends with the flag leaf.

[0091] Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin inthe leaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

[0092] Leaf-Sheath Pubescence: A rating of the pubescence of the leafsheath. Ratings are taken 1 to 2 weeks after pollination and scored aslight, medium, or heavy.

[0093] Leaf-Width: The average width of the primary ear leaf measured atits widest point.

[0094] LSS: Late season standability (values times 10 approximatepercent plants lodged in disease evaluation plots).

[0095] Moisture: The moisture of the grain at harvest.

[0096] On1: Ostrinia nubilalis 1st brood rating (1=resistant to9=susceptible).

[0097] On2: Ostrinia nubilalis 2nd brood rating (1=resistant to9=susceptible).

[0098] Relative Maturity: A maturity rating based on regressionanalysis. The regression analysis is developed by utilizing checkhybrids and their previously established day rating versus actualharvest moistures. Harvest moisture on the hybrid in question isdetermined and that moisture value is inserted into the regressionequation to yield a relative maturity.

[0099] Root Lodging: Root lodging is the percentage of plants that rootlodge. A plant is counted as root lodged if a portion of the plant leansfrom the vertical axis by approximately 30 degrees or more.

[0100] Seedling Color: Color of leaves at the 6 to 8 leaf stage.

[0101] Seedling Height: Plant height at the 6 to 8 leaf stage.

[0102] Seedling Vigor: A visual rating of the amount of vegetativegrowth on a 1 to 9 scale, where 1 equals best. The score is taken whenthe average entry in a trial is at the fifth leaf stage.

[0103] Selection Index: The selection index gives a single measure ofhybrid's worth based on information from multiple traits. One of thetraits that is almost always included is yield. Traits may be weightedaccording to the level of importance assigned to them.

[0104] Sr: Sphacelotheca reiliana rating is actual percent infection.

[0105] Stalk-Anthocyanin: A rating of the amount of anthocyaninpigmentation in the stalk. The stalk is rated 1 to 2 weeks afterpollination as absent, basal-weak, basal-strong, weak, or strong.

[0106] Stalk-Brace Root Color: The color of the brace roots observed 1to 2 weeks after pollination as green, red, or purple.

[0107] Stalk-Diameter: The average diameter of the lowest visibleinternode of the stalk.

[0108] Stalk-Ear Height: The average height of the ear measured from theground to the point of attachment of the ear shank of the top developedear to the stalk.

[0109] Stalk-Internode Direction: The direction of the stalk internodeobserved after pollination as straight or zigzag.

[0110] Stalk-Internode Length: The average length of the internode abovethe primary ear.

[0111] Stalk Lodging: The percentage of plants that did stalk lodge.Plants are counted as stalk lodged if the plant is broken over or offbelow the ear.

[0112] Stalk-Nodes With Brace Roots: The average number of nodes havingbrace roots per plant.

[0113] Stalk-Plant Height: The average height of the plant as measuredfrom the soil to the tip of the tassel.

[0114] Stalk-Tillers: The percent of plants that have tillers. A tilleris defined as a secondary shoot that has developed as a tassel capableof shedding pollen.

[0115] Staygreen: Staygreen is a measure of general plant health nearthe time of black layer formation (physiological maturity). It isusually recorded at the time the ear husks of most entries within atrial have turned a mature color. Scoring is on a 1 to 9 basis where 1equals best.

[0116] STR: Stalk rot rating (values represent severity rating of 1=25%of inoculated internode rotted to 9=entire stalk rotted and collapsed).

[0117] SVC: Southeastern Virus Complex (combination of Maize ChloroticDwarf Virus and Maize Dwarf Mosaic Virus) rating; numerical ratings arebased on a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

[0118] Tassel-Anther Color: The color of the anthers at 50% pollen shedscored as green-yellow, yellow, pink, red, or purple.

[0119] Tassel-Attitude: The attitude of the tassel after pollinationscored as open or compact.

[0120] Tassel-Branch Angle: The angle of an average tassel branch to themain stem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

[0121] Tassel-Branch Number: The average number of primary tasselbranches.

[0122] Tassel-Glume Band: The closed anthocyanin band at the base of theglume scored as present or absent.

[0123] Tassel-Glume Color: The color of the glumes at 50% shed scored asgreen, red, or purple.

[0124] Tassel-Length: The length of the tassel measured from the base ofthe bottom tassel branch to the tassel tip.

[0125] Tassel-Peduncle Length: The average length of the tasselpeduncle, measured from the base of the flag leaf to the base of thebottom tassel branch.

[0126] Tassel-Pollen Shed: A visual rating of pollen shed determined bytapping the tassel and observing the pollen flow of approximately fiveplants per entry. Rated on a 1 to 9 scale where 9=sterile, 1=mostpollen.

[0127] Tassel-Spike Length: The length of the spike measured from thebase of the top tassel branch to the tassel tip.

[0128] Test Weight: Weight of the grain in pounds for a given volume(bushel) adjusted to 15.5% moisture.

[0129] Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. OTHER DEFINITIONS

[0130] Allele: Any of one or more alternative forms of a gene locus, allof which alleles relate to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

[0131] Backcrossing: A process in which a breeder repeatedly crosseshybrid progeny back to one of the parents, for example, a firstgeneration hybrid (F₁) with one of the parental genotypes of the F₁hybrid.

[0132] Chromatography: A technique wherein a mixture of dissolvedsubstances are bound to a solid support followed by passing a column offluid across the solid support and varying the composition of the fluid.The components of the mixture are separated by selective elution.

[0133] Crossing: The pollination of a female flower of a corn plant,thereby resulting in the production of seed from the flower.

[0134] Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

[0135] Diploid: A cell or organism having two sets of chromosomes.

[0136] Electrophoresis: A process by which particles suspended in afluid or a gel matrix are moved under the action of an electrical field,and thereby separated according to their charge and molecular weight.This method of separation is well known to those skilled in the art andis typically applied to separating various forms of enzymes and of DNAfragments produced by restriction endonucleases.

[0137] Emasculate: The removal of plant male sex organs or theinactivation of the organs with a chemical agent or a cytoplasmic ornuclear genetic factor conferring male sterility.

[0138] Enzymes: Molecules that can act as catalysts in biologicalreactions.

[0139] F₁ Hybrid: The first generation progeny of the cross of twoplants.

[0140] Genetic Complement: An aggregate of nucleotide sequences, theexpression of which sequences defines the phenotype in corn plants, orcomponents of plants including cells or tissue.

[0141] Genotype: The genetic constitution of a cell or organism.

[0142] Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

[0143] Isozymes: Detectable variants of an enzyme, the variantscatalyzing the same reaction(s) but differing from each other, e.g., inprimary structure and/or electrophoretic mobility. The differencesbetween isozymes are under single gene, codominant control.Consequently, electrophoretic separation to produce band patterns can beequated to different alleles at the DNA level. Structural differencesthat do not alter charge cannot be detected by this method.

[0144] Isozyme typing profile: A profile of band patterns of isozymesseparated by electrophoresis that can be equated to different alleles atthe DNA level.

[0145] Linkage: A phenomenon wherein alleles on the same chromosome tendto segregate together more often than expected by chance if theirtransmission was independent.

[0146] Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

[0147] Metaxenia: An effect exerted on the phenotype of the embryo andassociated diploid tissues of a seed by the genotype contributed by thepollen.

[0148] HOI002: The corn plant variety from which seeds having ATCCAccession No. PTA-3788 were obtained, as well as plants grown from thoseseeds.

[0149] Phenotype: The detectable characteristics of a cell or organism,which characteristics are the manifestation of gene expression.

[0150] Quantitative Trait Loci (QTL): Genetic loci that contribute, atleast in part, certain numerically representable traits that are usuallycontinuously distributed.

[0151] Regeneration: The development of a plant from tissue culture.

[0152] SSR profile: A profile of simple sequence repeats used as geneticmarkers and scored by gel electrophoresis following PCR™ amplificationusing flanking oligonucleotide primers.

[0153] Self-pollination: The transfer of pollen from the anther to thestigma of the same plant.

[0154] Single Locus Converted (Conversion) Plant: Plants that aredeveloped by a plant breeding technique called backcrossing, whereinessentially all of the desired morphological and physiologicalcharacteristics of an inbred are recovered in addition to thecharacteristics conferred by the single locus transferred into theinbred via the backcrossing technique. A single locus may comprise onegene, or in the case of transgenic plants, one or more transgenesintegrated into the host genome at a single site (locus).

[0155] Substantially equivalent: The modifier “substantially equivalent”as used with respect to a first numerical value, such as that associatedwith a quantitative trait, for example, is used to include thosenumerical values that, when compared to the first numerical value, donot show statistical differences of their respective means.

[0156] Tissue Culture: A composition comprising isolated cells of thesame or a different type or a collection of such cells organized intoparts of a plant.

[0157] Transgene: A genetic sequence that has been introduced into thenuclear or chloroplast genome of a maize plant by a genetictransformation technique.

[0158] Xenia: An effect exerted on the phenotype of the endosperm of aseed by the genotype contributed by the pollen.

[0159] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

III. INBRED CORN PLANT HOI002

[0160] In accordance with one aspect of the present invention, there isprovided a novel inbred corn plant variety designated HOI002. Cornvariety HOI002 shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 1.HOI002 has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in HOI002.

[0161] Inbred corn plants can be reproduced by planting the seeds of theinbred corn plant HOI002, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

A. Phenotypic Description

[0162] In accordance with another aspect of the present invention, thereis provided a corn plant having the physiological and morphologicalcharacteristics of corn plant HOI002. A description of the physiologicaland morphological characteristics of corn plant HOI002 is given inTable 1. TABLE 1 Physiological and Morphological Traits for HOI002MATURITY: Days Heat Units From emergence to 50% of plants in silk: 801524 From emergence to 50% of plants in pollen 79 1496${{Heat}\quad {Units}\text{:}} = \frac{\begin{matrix}\lbrack {{{Max}.\quad {Temp}.\quad ( {\leq {86{^\circ}\quad {F.}}} )} +}  \\ {{{{Min}.\quad {Temp}}..}( {\geq {50{^\circ}\quad {F.}}} )} \rbrack\end{matrix} - 50}{2}$

[0163] PLANT:

[0164] Plant Height (to tassel tip): 206.0 cm

[0165] Ear Height (to base of top ear): 112.0 cm

[0166] Average number of Tillers: 0

[0167] Average Number of Ears per Stalk: 1

[0168] Anthocyanin of Brace Roots: Absent

[0169] LEAF:

[0170] Width of Ear Node Leaf: 9.0 cm

[0171] Length of Ear Node Leaf: 72.0 cm

[0172] Number of leaves above top ear: 5

[0173] Leaf Angle (from 2nd Leaf above ear at anthesis to Stalk aboveleaf): 27°

[0174] Leaf Color: Medium Green—Munsell Color Code 5 GY 4/4

[0175] Leaf Sheath Pubescence (Rate on scale from 1=none to 9=like peachfuzz): 2

[0176] Marginal Waves (Rate on scale from 1=none to 9=many): 4

[0177] Longitudinal Creases (Rate on scale from 1=none to 9=many): 5

[0178] TASSEL:

[0179] Number of Lateral Branches: 4

[0180] Branch Angle from Central Spike: 15°

[0181] Tassel Length (from top leaf collar to tassel top): 39.0 cm

[0182] Pollen Shed (Rate on scale from 0=male sterile to 9=heavy shed):6

[0183] Anther Color: Green-Yellow—Munsell Color Code 2.5 GY 8/10

[0184] Glume Color: Green—Munsell Color Code 5 GY 5/8

[0185] Bar Glumes: Absent

[0186] EAR: (Unhusked Data)

[0187] Silk Color (3 days after emergence): Light Green—Munsell ColorCode 2.5 GY 8/6

[0188] Fresh Husk Color (25 days after 50% silking): Light Green—MunsellColor Code 2.5

[0189] GY 7/8

[0190] Dry Husk Color (65 days after 50% silking): Buff—Munsell ColorCode 7.5 YR 7/4

[0191] Husk Extension: Short (ears exposed)

[0192] EAR: (Husked Ear Data)

[0193] Ear Length: 12.0 cm

[0194] Ear Diameter at mid-point: 42.0 mm

[0195] Ear Weight: 66.0 gm

[0196] Number of Kernel Rows: 18

[0197] Kernel Rows: Distinct

[0198] Shank Length: 10.0 cm

[0199] Ear Taper: Average

[0200] KERNEL: (Dried)

[0201] Kernel Length: 10.0 mm

[0202] Kernel Width: 7.0 mm

[0203] Kernel Thickness: 4.0 mm

[0204] Round Kernels (Shape Grade):

[0205] Aleurone Color Pattern: Homozygous

[0206] COB:

[0207] Cob Diameter at Mid-Point: 3.1

[0208] * These are typical values. Values may vary due to environment.Other values that are substantially equivalent are also within the scopeof the invention.

B. DEPOSIT INFORMATION

[0209] A representative deposit of 2500 seeds of the inbred corn varietydesignated HOI002 has been made with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. on Oct. 19,2001. Those deposited seeds have been assigned ATCC Accession No.PTA-3788. The deposit was made in accordance with the terms andprovisions of the Budapest Treaty relating to deposit of microorganismsand was made for a term of at least thirty (30) years and at least five(05) years after the most recent request for the furnishing of a sampleof the deposit is received by the depository, or for the effective termof the patent, whichever is longer, and will be replaced if it becomesnon-viable during that period.

IV. SINGLE LOCUS CONVERSIONS

[0210] When the term inbred corn plant is used in the context of thepresent invention, this also includes any single locus conversions ofthat inbred. The term single locus converted plant as used herein refersto those corn plants that are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of an inbred arerecovered in addition to the single locus transferred into the inbredvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theinbred. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental corn plants forthat inbred. The parental corn plant that contributes the locus or locifor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original inbred of interest (recurrent parent) is crossed to asecond inbred (nonrecurrent parent) that carries the single locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred locus from the nonrecurrent parent. The backcross processmay be accelerated by the use of genetic markers, such as SSR, RFLP, SNPor AFLP markers, to identify plants with the greatest genetic complementfrom the recurrent parent.

[0211] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original inbred. To accomplish this, a single locus of the recurrentinbred is modified or substituted with the desired locus from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0212] Many single locus traits have been identified that are notregularly selected for in the development of a new inbred but that canbe improved by backcrossing techniques. Single locus traits may or maynot be transgenic; examples of these traits include, but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus, but may be inherited through the cytoplasm. Some knownexceptions to this are genes for male sterility, some of which areinherited cytoplasmically, but still act as single locus traits. Anumber of exemplary single locus traits are described in, for example,PCT Application WO 95/06128, the disclosure of which is specificallyincorporated herein by reference.

[0213] Examples of genes conferring male sterility include thosedisclosed in U.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat.No. 4,654,465, U.S. Pat. No 5,625,132, and U.S. Pat. No. 4,727,219, eachof the disclosures of which are specifically incorporated herein byreference in their entirety. A particularly useful type of malesterility gene is one that can be induced by exposure to a chemicalagent, for example, a herbicide (U.S. patent Ser. No. 08/927,368, filedSept. 11, 1997, the disclosure of which is specifically incorporatedherein by reference in its entirety). Both inducible and non-induciblemale sterility genes can increase the efficiency with which hybrids aremade, in that they eliminate the need to physically emasculate the cornplant used as a female in a given cross.

[0214] Where one desires to employ male-sterility systems with a cornplant in accordance with the invention, it may be beneficial to alsoutilize one or more male-fertility restorer genes. For example, wherecytoplasmic male sterility (CMS) is used, hybrid seed productionrequires three inbred lines: (1) a cytoplasmically male-sterile linehaving a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm,which is isogenic with the CMS line for nuclear genes (“maintainerline”); and (3) a distinct, fertile inbred with normal cytoplasm,carrying a fertility restoring gene (“restorer” line). The CMS line ispropagated by pollination with the maintainer line, with all of theprogeny being male sterile, as the CMS cytoplasm is derived from thefemale parent. These male sterile plants can then be efficientlyemployed as the female parent in hybrid crosses with the restorer line,without the need for physical emasculation of the male reproductiveparts of the female parent.

[0215] The presence of a male-fertility restorer gene results in theproduction of fully fertile F₁ hybrid progeny. If no restorer gene ispresent in the male parent, male-sterile hybrids are obtained. Suchhybrids are useful where the vegetative tissue of the corn plant isutilized, e.g., for silage, but in most cases, the seeds will be deemedthe most valuable portion of the crop, so fertility of the hybrids inthese crops must be restored. Therefore, one aspect of the currentinvention concerns the inbred corn plant HOI0012 comprising a singlegene capable of restoring male fertility in an otherwise male-sterileinbred or hybrid plant. Examples of male-sterility genes andcorresponding restorers that could be employed with the inbred of theinvention are well known to those of skill in the art of plant breedingand are disclosed in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat.No. 5,689,041; U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

[0216] Direct selection may be applied where a single locus acts as adominant trait. An example of a dominant trait is the herbicideresistance trait. For this selection process, the progeny of the initialcross are sprayed with the herbicide prior to the backcrossing. Thespraying eliminates any plants that do not have the desired herbicideresistance characteristic, and only those plants that have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

[0217] Many useful single locus traits are those that are introduced bygenetic transformation techniques. Methods for the genetictransformation of maize are known to those of skill in the art. Forexample, methods that have been described for the genetic transformationof maize include electroporation (U.S. Pat. No. 5,384,253),electrotransformation (U.S. Pat. No. 5,371,003), microprojectilebombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat.No. 5,538,880; and PCT Publication WO 95/06128), Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,591,616 and E.P. Publication EP672752),direct DNA uptake transformation of protoplasts (Omirulleh et al., 1993)and silicon carbide fiber-mediated transformation (U.S. Pat. No.5,302,532 and U.S. Pat. No. 5,464,765).

[0218] A type of single locus trait that can be introduced by genetictransformation (U.S. Pat. No. 5,554,798) and has particular utility is agene that confers resistance to the herbicide glyphosate. Glyphosateinhibits the action of the enzyme EPSPS, which is active in thebiosynthetic pathway of aromatic amino acids. Inhibition of this enzymeleads to starvation for the amino acids phenylalanine, tyrosine, andtryptophan and secondary metabolites derived therefrom. Mutants of thisenzyme are available that are resistant to glyphosate. For example, U.S.Pat. No. 4,535,060 describes the isolation of EPSPS mutations thatconfer glyphosate resistance upon organisms having the Salmonellatyphimurium gene for EPSPS, aroA. A mutant EPSPS gene having similarmutations has also been cloned from Zea mays. The mutant gene encodes aprotein with amino acid changes at residues 102 and 106 (PCT PublicationWO 97/04103). When a plant comprises such a gene, a herbicide resistantphenotype results.

[0219] Plants having inherited a transgene comprising a mutated EPSPSgene may, therefore, be directly treated with the herbicide glyphosatewithout the result of significant damage to the plant. This phenotypeprovides farmers with the benefit of controlling weed growth in a fieldof plants having the herbicide resistance trait by application of thebroad spectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields whereglyphosate resistant corn plants are grown. The herbicide applicationrates may typically range from 4 ounces of ROUNDUP™ to 256 ouncesROUNDUP™ per acre. More preferably, about 16 ounces to about 64 ouncesper acre of ROUNDUP™ may be applied to the field. However, theapplication rate may be increased or decreased as needed, based on theabundance and/or type of weeds being treated. Additionally, depending onthe location of the field and weather conditions, which will influenceweed growth and the type of weed infestation, it may be desirable toconduct further glyphosate treatments. The second glyphosate applicationwill also typically comprise an application rate of about 16 ounces toabout 64 ounces of ROUNDUP™ per acre treated. Again, the treatment ratemay be adjusted based on field conditions. Such methods of applicationof herbicides to agricultural crops are well known in the art and aresummarized in general in Anderson (1983).

[0220] Alternatively, more than one single locus trait may beintrogressed into an elite inbred by the method of backcross conversion.A selectable marker gene and a gene encoding a protein that confers atrait of interest may be simultaneously introduced into a maize plant asa result of genetic transformation. Usually one or more introduced geneswill integrate into a single chromosome site in the host cell's genome.For example, a selectable marker gene encoding phosphinothricin acetyltransferase (PPT) (e.g., a bar gene) and conferring resistance to theactive ingredient in some herbicides by inhibiting glutamine synthetase,and a gene encoding an endotoxin from Bacillus thuringiensis (Bt) andconferring resistance to particular classes of insects, e.g.,lepidopteran insects, in particular the European Corn Borer, may besimultaneously introduced into a host genome. Furthermore, through theprocess of backcross conversion more than one transgenic trait may betransferred into an elite inbred.

[0221] The waxy characteristic is an example of a recessive trait. Inthis example, the progeny resulting from the first backcross generation(BC1) must be grown and selfed. A test is then run on the selfed seedfrom the BC1 plant to determine which BC1 plants carried the recessivegene for the waxy trait. In other recessive traits additional progenytesting, for example growing additional generations such as the BC1S1,may be required to determine which plants carry the recessive gene.

V. ORIGIN AND BREEDING HISTORY OF AN EXEMPLARY SINGLE LOCUS CONVERTEDPLANT

[0222] Methods for the preparation of single gene converted plants areknown to those of skill in the art. An example of such a single geneconverted plant is 85DGD1 MLms, which is a single locus conversion ofthe corn line 85DGD1 to cytoplasmic male sterility. The methods used toconvert this line were described in U.S. Pat. No. 6,175,063, thedisclosure of which is incorporated herein in the entirety, and are setforth herein below. As described in U.S. Pat. No. 6,175,063, 85DGD1 MLmswas derived using backcross methods. In particular, the line designated85DGD1 was used as the recurrent parent and MLms, a germplasm sourcecarrying ML cytoplasmic sterility, was used as the nonrecurrent parent.The breeding history of the single locus converted inbred 85DGD1 MLmscan be summarized as follows: Hawaii Nurseries Planting Date Apr. 02,1992 Made up S-O: Female row 585 male row 500 Hawaii Nurseries PlantingDate Jul. 15, 1992 S-O was grown and plants were backcrossed times85DGD1 (rows 444 ′ 443) Hawaii Nurseries Planting Date Nov. 18, 1992Bulked seed of the BC1 was grown and backcrossed times 85DGD1 (rows V3-27 ′ V3-26) Hawaii Nurseries Planting Date Apr. 02, 1993 Bulked seed ofthe BC2 was grown and backcrossed times 85DGD1 (rows 37 36) HawaiiNurseries Planting Date Jul. 14, 1993 Bulked seed of the BC3 was grownand backcrossed times 85DGD1 (rows 99 ′ 98) Hawaii Nurseries PlantingDate Oct. 28, 1993 Bulked seed of BC4 was grown and backcrossed times85DGD1 (rows KS- 63 ′ KS-62) Summer 1994 A single ear of the BC5 wasgrown and backcrossed times 85DGD1 (MC94-822 MC94-822-7) Winter 1994Bulked seed of the BC6 was grown and backcrossed times 85DGD1 (3Q-1 ′3Q- 2) Summer 1995 Seed of the BC7 was bulked and named 85DGD1 MLms.

VI. TISSUE CULTURES AND IN VITRO REGENERATION OF CORN PLANTS

[0223] A further aspect of the invention relates to tissue cultures ofthe corn plant designated HOI002. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

VII. TASSEL/ANTHER CULTURE

[0224] Tassels contain anthers that in turn enclose microspores.Microspores develop into pollen. For anther/microspore culture, iftassels are the plant composition, they are preferably selected at astage when the microspores are uninucleate, that is, include only one,rather than 2 or 3 nuclei. Methods to determine the correct stage arewell known to those skilled in the art and include mitramycinfluorescent staining (Pace et al., 1987), trypan blue (preferred) andacetocarmine squashing. The mid-uninucleate microspore stage has beenfound to be the developmental stage most responsive to the subsequentmethods disclosed to ultimately produce plants.

[0225] Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

[0226] Although not required, when tassels are employed as the plantorgan, it is generally preferred to sterilize their surface. Followingsurface sterilization of the tassels, for example, with a solution ofcalcium hypochloride, the anthers are removed from about 70 to 150spikelets (small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

[0227] When one elects to employ tassels directly, tassels arepreferably pretreated at a cold temperature for a predefined time,preferably at 10° C. for about 4 days. After pretreatment of a wholetassel at a cold temperature, dissected anthers are further pretreatedin an environment that diverts microspores from their developmentalpathway. The function of the preculture medium is to switch thedevelopmental program from one of pollen development to that ofembryoid/callus development. An embodiment of such an environment in theform of a preculture medium includes a sugar alcohol, for examplemannitol or sorbitol, inositol or the like. An exemplary synergisticcombination is the use of mannitol at a temperature of about 10° C. fora period ranging from about 10 to 14 days. In a preferred embodiment, 3ml of 0.3 M mannitol combined with 50 mg/l of ascorbic acid, silvernitrate, and colchicine is used for incubation of anthers at 10° C. forbetween 10 and 14 days. Another embodiment is to substitute sorbitol formannitol. The colchicine produces chromosome doubling at this earlystage. The chromosome doubling agent is preferably only present at thepreculture stage.

[0228] It is believed that the mannitol or other similar carbonstructure or environmental stress induces starvation and functions toforce microspores to focus their energies on entering developmentalstages. The cells are unable to use, for example, mannitol as a carbonsource at this stage. It is believed that these treatments confuse thecells causing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴microspores, have resulted from using these methods.

[0229] In embodiments where microspores are obtained from anthers,microspores can be released from the anthers into an isolation mediumfollowing the mannitol preculture step. One method of release is bydisruption of the anthers, for example, by chopping the anthers intopieces with a sharp instrument, such as a razor blade, scalpel, orWaring blender. The resulting mixture of released microspores, antherfragments, and isolation medium are then passed through a filter toseparate microspores from anther wall fragments. An embodiment of afilter is a mesh, more specifically, a nylon mesh of about 112 mm poresize. The filtrate that results from filtering the microspore-containingsolution is preferably relatively free of anther fragments, cell walls,and other debris.

[0230] In a preferred embodiment, isolation of microspores isaccomplished at a temperature below about 25° C. and preferably, at atemperature of less than about 15° C. Preferably, the isolation media,dispersing tool (e.g., razor blade), funnels, centrifuge tubes, anddispersing container (e.g., petri dish) are all maintained at thereduced temperature during isolation. The use of a precooled dispersingtool to isolate maize microspores has been reported (Gaillard et al.,1991).

[0231] Where appropriate and desired, the anther filtrate is then washedseveral times in isolation medium. The purpose of the washing andcentrifugation is to, eliminate any toxic compounds that are containedin the non-microspore part of the filtrate and are created by thechopping process. The centrifugation is usually done at decreasing spinspeeds, for example, 1000, 750, and finally 500 rpms. The result of theforegoing steps is the preparation of a relatively pure tissue culturesuspension of microspores that are relatively free of debris and antherremnants.

[0232] To isolate microspores, an isolation media is preferred. Anisolation media is used to separate microspores from the anther wallswhile maintaining their viability and embryogenic potential. Anillustrative embodiment of an isolation media includes a 6% sucrose ormaltose solution combined with an antioxidant such as 50 mg/l ofascorbic acid, 0.1 mg/l biotin, and 400 mg/l of proline, combined with10 mg/l of nicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, thebiotin and proline are omitted.

[0233] An isolation media preferably has a higher antioxidant levelwhere it is used to isolate microspores from a donor plant (a plant fromwhich a plant composition containing a microspore is obtained) that isfield grown in contrast to greenhouse grown. A preferred level ofascorbic acid in an isolation medium is from about 50 mg/l to about 125mg/l and, more preferably, from about 50 mg/l to about 100 mg/l.

[0234] One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. The microsporesuspension is layered onto a support, for example by pipetting. Thereare several types of supports that are suitable and are within the scopeof the invention. An illustrative embodiment of a solid support is aTRANSWELL® culture dish. Another embodiment of a solid support fordevelopment of the microspores is a bilayer plate wherein liquid mediais on top of a solid base. Other embodiments include a mesh or amillipore filter. Preferably, a solid support is a nylon mesh in theshape of a raft. A raft is defined as an approximately circular supportmaterial that is capable of floating slightly above the bottom of atissue culture vessel, for example, a petri dish, of about a 60 or 100mm size, although any other laboratory tissue culture vessel willsuffice. In an illustrative embodiment, a raft is about 55 mm indiameter.

[0235] Culturing isolated microspores on a solid support, for example,on a 10 mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petridish, prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent). The charcoal is believed to absorb toxic wastes andintermediaries. The solid medium allows embryoids to mature.

[0236] The liquid medium passes through the mesh while the microsporesare retained and supported at the medium-air interface. The surfacetension of the liquid medium in the petri dish causes the raft to float.The liquid is able to pass through the mesh; consequently, themicrospores stay on top. The mesh remains on top of the total volume ofliquid medium. An advantage of the raft is to permit diffusion ofnutrients to the microspores. Use of a raft also permits transfer of themicrospores from dish to dish during subsequent subculture with minimalloss, disruption, or disturbance of the induced embryoids that aredeveloping. The rafts represent an advantage over the multi-welledTRANSWELL® plates, which are commercially available from COSTAR, in thatthe commercial plates are expensive. Another disadvantage of theseplates is that to achieve the serial transfer of microspores tosubsequent media, the membrane support with cells must be peeled off theinsert in the wells. This procedure does not produce as good a yield noras efficient transfers, as when a mesh is used as a vehicle for celltransfer.

[0237] The culture vessels can be further defined as either (1) abilayer 60 mm petri plate wherein the bottom 2 ml of medium aresolidified with 0.7% agarose overlaid with 1 mm of liquid containing themicrospores; (2) a nylon mesh raft wherein a wafer of nylon is floatedon 1.2 ml of medium and 1 ml of isolated microspores is pipetted on top;or (3) TRANSWELL® plates wherein isolated microspores are pipetted ontomembrane inserts that support the microspores at the surface of 2 ml ofmedium.

[0238] After the microspores have been isolated, they are cultured in alow strength anther culture medium until about the 50 cell stage whenthey are subcultured onto an embryoid/callus maturation medium. Mediumis defined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, and growth regulators. A solidifying agent isoptional. A preferred embodiment of such a media is referred to as “Dmedium,” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

[0239] In an illustrative embodiment, 1 ml of isolated microspores arepipetted onto a 10 mm nylon raft and the raft is floated on 1.2 ml ofmedium “D,” containing sucrose or preferably maltose. Both calli andembryoids can develop. Calli are undifferentiated aggregates of cells.Type I is a relatively compact, organized, and slow growing callus. TypeII is a soft, friable, and fast-growing one. Embryoids are aggregatesexhibiting some embryo-like structures. The embryoids are preferred forsubsequent steps to regenerating plants. Culture medium “D” is anembodiment of medium that follows the isolation medium and replaces it.Medium “D” promotes growth to an embryoid/callus. This medium comprises6N1 salts at ⅛ the strength of a basic stock solution (major components)and minor components, plus 12% sucrose, or preferably 12% maltose, 0.1mg/l B1, 0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5 mg/l silvernitrate. Silver nitrate is believed to act as an inhibitor to the actionof ethylene. Multi-cellular structures of approximately 50 cells eachgenerally arise during a period of 12 days to 3 weeks. Serial transferafter a two week incubation period is preferred.

[0240] After the petri dish has been incubated for an appropriate periodof time, preferably two weeks in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media that promote embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12% sugar(sucrose, maltose, or a combination thereof), 0.5% activated charcoal,400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percentGELRITE™ (solidifying agent) and is capable of promoting the maturationof the embryoids. Higher quality embryoids, that is, embryoids thatexhibit more organized development, such as better shoot meristemformation without precocious germination, were typically obtained withthe transfer to full strength medium compared to those resulting fromcontinuous culture using only, for example, the isolated microsporeculture (IMC) Medium “D.” The maturation process permits the pollenembryoids to develop further in route toward the eventual regenerationof plants. Serial transfer occurs to full strength solidified 6N1 mediumusing either the nylon raft, the TRANSWELL® membrane, or bilayer plates,each one requiring the movement of developing embryoids to permitfurther development into physiologically more mature structures. In anespecially preferred embodiment, microspores are isolated in anisolation media comprising about 6% maltose, cultured for about twoweeks in an embryoid/calli induction medium comprising about 12% maltoseand then transferred to a solid medium comprising about 12% sucrose.

[0241] At the point of transfer of the raft, after about two weeks ofincubation, embryoids exist on a nylon support. The purpose oftransferring the raft with the embryoids to a solidified medium afterthe incubation is to facilitate embryo maturation. Mature embryoids atthis point are selected by visual inspection indicated by zygoticembryo-like dimensions and structures and are transferred to the shootinitiation medium. It is preferred that shoots develop before roots, orthat shoots and roots develop concurrently. If roots develop beforeshoots, plant regeneration can be impaired. To produce solidified media,the bottom of a petri dish of approximately 100 mm is covered with about30 ml of 0.2% GELRITE™ solidified medium. A sequence of regenerationmedia are used for whole plant formation from the embryoids.

[0242] During the regeneration process, individual embryoids are inducedto form plantlets. The number of different media in the sequence canvary depending on the specific protocol used. Finally, a rooting mediumis used as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

[0243] Plants have been produced from isolated microspore cultures bythe methods disclosed herein, including self-pollinated plants. The rateof embryoid induction was much higher with the synergistic preculturetreatment consisting of a combination of stress factors, including acarbon source that can be capable of inducing starvation, a coldtemperature, and colchicine, than has previously been reported. Anillustrative embodiment of the synergistic combination of treatmentsleading to the dramatically improved response rate compared to priormethods, is a temperature of about 10° C., mannitol as a carbon source,and 0.05% colchicine.

[0244] The inclusion of ascorbic acid, an anti-oxidant, in the isolationmedium is preferred for maintaining good microspore viability. However,there seems to be no advantage to including mineral salts in theisolation medium. The osmotic potential of the isolation medium wasmaintained optimally with about 6% sucrose, although a range of 2% to12% is within the scope of this invention.

[0245] In an embodiment of the embryoid/callus organizing media, mineralsalts concentration in IMC Culture Media “D” is (⅛x), the concentrationthat is used also in anther culture medium. The 6N1 salts majorcomponents have been modified to remove ammonium nitrogen. Osmoticpotential in the culture medium is maintained with about 12% sucrose andabout 400 mg/l proline. Silver nitrate (0.5 mg/l) was included in themedium to modify ethylene activity. The preculture media is furthercharacterized by having a pH of about 5.7 to 6.0. Silver nitrate andvitamins do not appear to be crucial to this medium but do improve theefficiency of the response.

[0246] Whole anther cultures can also be used in the production ofmonocotyledonous plants from a plant culture system. There are somebasic similarities of anther culture methods and microspore culturemethods with regard to the media used. A difference from isolatedmicrospore cultures is that undisrupted anthers are cultured, so that asupport, e.g., a nylon mesh support, is not needed. The first step indeveloping the anther cultures is to incubate tassels at a coldtemperature. A cold temperature is defined as less than about 25° C.More specifically, the incubation of the tassels is preferably performedat about 10° C. A range of 8 to 14° C. is also within the scope of theinvention. The anthers are then dissected from the tassels, preferablyafter surface sterilization using forceps, and placed on solidifiedmedium. An example of such a medium is designated 6N1-TGR-P4.

[0247] The anthers are then treated with environmental conditions thatare combinations of stresses that are capable of diverting microsporesfrom gametogenesis to embryogenesis. It is believed that the stresseffect of sugar alcohols in the preculture medium, for example,mannitol, is produced by inducing starvation at the predefinedtemperature. In one embodiment, the incubation pretreatment is for about14 days at 10° C. It was found that treating the anthers in additionwith a carbon structure, an illustrative embodiment being a sugaralcohol, preferably mannitol, produces dramatically higher antherculture response rates as measured by the number of eventuallyregenerated plants, than by treatment with either cold treatment ormannitol alone. These results are particularly surprising in light ofteachings that cold is better than mannitol for these purposes, and thatwarmer temperatures interact with mannitol better.

[0248] To incubate the anthers, they are floated on a preculture mediumthat diverts the microspores from gametogenesis, preferably on amannitol carbon structure, more specifically, 0.3 M of mannitol plus 50mg/l of ascorbic acid. Three milliliters is about the total amount in adish, for example, a tissue culture dish, more specifically, a 60 mmpetri dish. Anthers are isolated from about 120 spikelets for one dishyields about 360 anthers.

[0249] Chromosome doubling agents can be used in the preculture mediafor anther cultures. Several techniques for doubling chromosome number(Jensen, 1974; Wan et al., 1989) have been described. Colchicine is oneof the doubling agents. However, developmental abnormalities arisingfrom in vitro cloning are further enhanced by colchicine treatments, andprevious reports indicated that colchicine is toxic to microspores. Theaddition of colchicine in increasing concentrations during mannitolpretreatment prior to anther culture and microspore culture has achievedimproved percentages.

[0250] An illustrative embodiment of the combination of a chromosomedoubling agent and preculture medium is one that contains colchicine. Ina specific embodiment, the colchicine level is preferably about 0.05%.The anthers remain in the mannitol preculture medium with the additivesfor about 10 days at 10° C. Anthers are then placed on maturation media,for example, that designated 6N1-TGR-P4, for 3 to 6 weeks to induceembryoids. If the plants are to be regenerated from the embryoids, shootregeneration medium is employed, as in the isolated microspore proceduredescribed in the previous sections. Other regeneration media can be usedsequentially to complete regeneration of whole plants.

[0251] The anthers are then exposed to embryoid/callus promoting medium,for example, that designated 6N1-TGR-P4, to obtain callus or embryoids.The embryoids are recognized visually by identification ofembryonic-like structures. At this stage, the embryoids are transferredprogressively through a series of regeneration media. In an illustrativeembodiment, the shoot initiation medium comprises BAP(6-benzyl-amino-purine) and NAA (naphthalene acetic acid). Regenerationprotocols for isolated microspore cultures and anther cultures aresimilar.

VIII. ADDITIONAL TISSUE CULTURES AND REGENERATION

[0252] The present invention contemplates a corn plant regenerated froma tissue culture of the inbred maize plant HOI002, or of a hybrid maizeplant produced by crossing HOI002. As is well known in the art, tissueculture of corn can be used for the in vitro regeneration of a cornplant. By way of example, a process of tissue culturing and regenerationof corn is described in European Patent Application 0 160 390, thedisclosure of which is incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes (1982) andDuncan et al. (1985). The study by Duncan et al. (1985) indicates that97 percent of cultured plants produced calli capable of regeneratingplants. Subsequent studies have shown that both inbreds and hybridsproduced 91% regenerable calli that produced plants.

[0253] Other studies indicate that non-traditional tissues are capableof producing somatic embryogenesis and plant regeneration (Songstad etal., 1988; Rao et al., 1986; Conger et al., 1987; the disclosures ofwhich are incorporated herein by reference). Regenerable cultures,including Type I and Type II cultures, may be initiated from immatureembryos using methods described in, for example, PCT Application WO95/06128, the disclosure of which is incorporated herein by reference inits entirety.

[0254] Briefly, by way of example, to regenerate a plant of thisinvention, cells are selected following growth in culture. Whereemployed, cultured cells are preferably grown either on solid supportsor in the form of liquid suspensions as set forth above. In eitherinstance, nutrients are provided to the cells in the form of media, andenvironmental conditions are controlled. There are many types of tissueculture media comprising amino acids, salts, sugars, hormones, andvitamins. Most of the media employed to regenerate inbred and hybridplants have some similar components; the media differ in the compositionand proportions of their ingredients depending on the particularapplication envisioned. For example, various cell types usually grow inmore than one type of media, but exhibit different growth rates anddifferent morphologies, depending on the growth media. In some media,cells survive but do not divide. Various types of media suitable forculture of plant cells have been previously described and discussedabove.

[0255] An exemplary embodiment for culturing recipient corn cells insuspension cultures includes using embryogenic cells in Type II(Armstrong and Green, 1985; Gordon-Kamm et al., 1990) callus, selectingfor small (10 to 30 mm) isodiametric, cytoplasmically dense cells,growing the cells in suspension cultures with hormone containing media,subculturing into a progression of media to facilitate development ofshoots and roots, and finally, hardening the plant and readying itmetabolically for growth in soil.

[0256] Meristematic cells (i.e., plant cells capable of continual celldivision and characterized by an undifferentiated cytologicalappearance, normally found at growing points or tissues in plants suchas root tips, stem apices, lateral buds, etc.) can be cultured (U.S.Pat. No. 5,736,369, the disclosure of which is specifically incorporatedherein by reference).

[0257] Embryogenic calli are produced essentially as described in PCTApplication WO 95/06128. Specifically, inbred plants or plants fromhybrids produced from crossing an inbred of the present invention withanother inbred are grown to flowering in a greenhouse. Explants from atleast one of the following F₁ tissues: the immature tassel tissue,intercalary meristems and leaf bases, apical meristems, immature earsand immature embryos are placed in an initiation medium that contain MSsalts, supplemented with thiamine, agar, and sucrose. Cultures areincubated in the dark at about 23° C. All culture manipulations andselections are performed with the aid of a dissecting microscope.

[0258] After about 5 to 7 days, cellular outgrowths are observed fromthe surface of the explants. After about 7 to 21 days, the outgrowthsare subcultured by placing them into fresh medium of the samecomposition. Some of the intact immature embryo explants are placed onfresh medium. Several subcultures later (after about 2 to 3 months)enough material is present from explants for subdivision of theseembryogenic calli into two or more pieces.

[0259] Callus pieces from different explants are not mixed. Afterfurther growth and subculture (about 6 months after embryogenic callusinitiation), there are usually between 1 and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Material known fromexperience to lack the capacity for sustained growth is also discarded(translucent, watery, embryogenic structures). Structures with a firmconsistency resembling at least in part the scutelum of the in vivoembryo are selected.

[0260] The callus is maintained on agar-solidified MS or N6-type media.A preferred hormone is 2,4-D. A second preferred hormone is dicamba.Visual selection of embryo-like structures is done to obtainsubcultures. Transfer of material other than that displaying embryogenicmorphology results in loss of the ability to recover whole plants fromthe callus.

[0261] Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS or N6Medium containing 2,4-D or dicamba. The calli in medium are incubated atabout 27° C. on a gyrotary shaker in the dark or in the presence of lowlight. The resultant suspension culture is transferred about once everythree to seven days, preferably every three to four days, by takingabout 5 to 10 ml of the culture and introducing this inoculum into freshmedium of the composition listed above (PCT Application WO 95/06128).

[0262] For regeneration of type I or type II callus, callus istransferred to a solidified culture medium that includes a lowerconcentration of 2,4-D or other auxins than is present in culture mediumused for callus maintenance (PCT Application WO 95/06128, specificallyincorporated herein by reference). Other hormones that can be used inregeneration media include dicamba, NAA, ABA, BAP, and 2-NCA.Regeneration of plants is completed by the transfer of mature andgerminating embryos to a hormone-free medium, followed by the transferof developed plantlets to soil and growth to maturity. Plantregeneration is described in PCT Application WO 95/06128.

[0263] Cells from the meristem or cells fated to contribute to themeristem of a cereal plant embryo at the early proembryo, mid proembryo,late proembryo, transitional or early coleoptilar stage may be culturedso as to produce a proliferation of shoots or multiple meristems fromwhich fertile plants may be regenerated. Alternatively, cells from themeristem or cells fated to contribute to the meristem of a cereal plantimmature ear or tassel may be cultured so as to produce a proliferationof shoots or multiple meristems from which fertile plants may beregenerated (U.S. Pat. No. 5,736,369).

[0264] Progeny of any generation are produced by taking pollen andselfing, backcrossing, or sibling crossing regenerated plants by methodswell known to those skilled in the arts. Seeds are collected from theregenerated plants. Alternatively, progeny of any generation may beproduced by pollinating a regenerated plant with its own pollen orpollen of a second maize plant. Using the methods described herein,tissue cultures and immature or mature plant tissues may be used asrecipient cell cultures for the process of genetic transformation.

IX. PROCESSES OF PREPARING CORN PLANTS AND THE CORN PLANTS PRODUCED BYSUCH CROSSES

[0265] The present invention also provides a process of preparing anovel corn plant and a corn plant produced by such a process. Inaccordance with such a process, a first parent corn plant is crossedwith a second parent corn plant wherein at least one of the first andsecond corn plants is the inbred corn plant HOI002. An important aspectof this process is that it can be used for the development of novelinbred lines. For example, the inbred corn plant HOI002 could be crossedto any second plant, and the resulting hybrid progeny each selfed forabout 5 to 7 or more generations, thereby providing a large number ofdistinct, pure-breeding inbred lines. These inbred lines could then becrossed with other inbred or non-inbred lines and the resulting hybridprogeny analyzed for beneficial characteristics. In this way, novelinbred lines conferring desirable characteristics could be identified.

[0266] In selecting a second plant to cross with HOI002 for the purposeof developing novel inbred lines, it will typically be desired choosethose plants that either themselves exhibit one or more selecteddesirable characteristics or that exhibit the desired characteristic(s)when in hybrid combination. Examples of potentially desiredcharacteristics include greater yield, better stalks, better roots,resistance to insecticides, herbicides, pests, and disease, tolerance toheat and drought, reduced time to crop maturity, better agronomicquality, higher nutritional value, and uniformity in germination times,stand establishment, growth rate, maturity, and fruit size.Alternatively, the inbred variety HOI002 may be crossed with a second,different inbred plant for the purpose of producing hybrid seed that issold to farmers for planting in commercial production fields. In thiscase, a second inbred variety is selected that confers desirablecharacteristics when in hybrid combination with the first inbred line.

[0267] Corn plants (Zea mays L.) can be crossed by either natural ormechanical techniques. Natural pollination occurs in corn when windblows pollen from the tassels to the silks that protrude from the topsof the recipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand.

[0268] In a preferred embodiment, crossing comprises the steps of:

[0269] (a) planting in pollinating proximity seeds of a first and asecond parent corn plant, and preferably, seeds of a first inbred cornplant and a second, distinct inbred corn plant;

[0270] (b) cultivating or growing the seeds of the first and secondparent corn plants into plants that bear flowers;

[0271] (c) emasculating flowers of either the first or second parentcorn plant, i.e., treating the flowers so as to prevent pollenproduction, or alternatively, using as the female parent a male sterileplant, thereby providing an emasculated parent corn plant;

[0272] (d) allowing natural cross-pollination to occur between the firstand second parent corn plants;

[0273] (e) harvesting seeds produced on the emasculated parent cornplant; and, where desired,

[0274] (f) growing the harvested seed into a corn plant, preferably, ahybrid corn plant.

[0275] Parental plants are typically planted in pollinating proximity toeach other by planting the parental plants in alternating rows, inblocks or in any other convenient planting pattern. Where the parentalplants differ in timing of sexual maturity, it may be desired to plantthe slower maturing plant first, thereby ensuring the availability ofpollen from the male parent during the time at which silks on the femaleparent are receptive to pollen. Plants of both parental parents arecultivated and allowed to grow until the time of flowering.Advantageously, during this growth stage, plants are in general treatedwith fertilizer and/or other agricultural chemicals as consideredappropriate by the grower.

[0276] At the time of flowering, in the event that plant HOI002 isemployed as the male parent, the tassels of the other parental plant areremoved from all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factor thatcauses resistance to the emasculating effects of the chemical agent.Gametocides affect processes or cells involved in the development,maturation or release of pollen. Plants treated with such gametocidesare rendered male sterile, but typically remain female fertile. The useof chemical gametocides is described, for example, in U.S. Pat. No.4,936,904, the disclosure of which is specifically incorporated hereinby reference in its entirety. Furthermore, the use of Roundup herbicidein combination with glyphosate tolerant maize plants to produce malesterile corn plants is disclosed in U.S. patent application Ser. No.08/927,368 and PCT Publication WO 98/44140.

[0277] Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

[0278] Both parental inbred plants of corn may be allowed to continue togrow until maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

[0279] Alternatively, in another embodiment of the invention, both firstand second parent corn plants can come from the same inbred corn plant,i.e., from the inbred designated HOI002. Thus, any corn plant producedusing a process of the present invention and inbred corn plant HOI002,is contemplated by the current inventor. As used herein, crossing canmean selfing, backcrossing, crossing to another or the same inbred,crossing to populations, and the like. All corn plants produced usingthe inbred corn plant HOI002 as a parent are, therefore, within thescope of this invention.

[0280] The utility of the inbred plant HOI002 also extends to crosseswith other species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Of these, Zea and Tripsacum, are most preferred. Potentiallysuitable for crosses with HOI002 can also be the various varieties ofgrain sorghum, Sorghum bicolor (L.) Moench.

A. Utilization of HOI002 as a Pollinator for Enhancing Kernel QualityTraits

[0281] One advantage of the variety of the invention is that itpossesses markedly enhanced quality grain traits relative to typicalprior varieties. These traits can be conferred through crossing for theproduction of kernels and plants grown therefrom with enhanced qualitytraits. Such enhanced traits may include, for example, elevated oilcontent, elevated protein content, improved oil quality, enhancedoxidative stability of the oil, reduced polyunsaturated fatty acids inthe oil, elevated oleic acid content, improved nutritional quality ofthe protein and improved functional properties of the starch, as well asother traits. In particular embodiments of the invention, plantsobtained can comprise one or more enhanced quality traits selected fromthe group consisting of: oil content in excess of 6% of the seed drymatter, protein content in excess of 10% of the seed dry matter, oleicacid content in excess of 35% of the total fatty acids of the oil,lysine content in excess of 0.32% of the seed dry matter and tryptophancontent in excess of 0.08% of the seed dry matter. Such enhanced qualitytraits may be conferred in hybrid combination.

[0282] The variety HOI002 can also be used to confer enhanced qualitygrain traits by way of the xenia and/or metaxenia effect, in whichquality traits of pollen affect the grain resulting from thepollinating. One method for exploiting the effect involves planting inpollinating proximity in a field corn seed of the variety HOI002 andcorn seed of an agronomically elite variety. By allowing these plants togrow together, pollen of the variety HOI002 becomes available topollinate the female flowers of the agronomically elite variety. Thepollinating confers enhanced quality grain traits upon the kernelsproduced by the pollinating, but the yield of grain will otherwiseapproach that normally obtained using the agronomically elite “female”parent. Preferably, the agronomically elite plant is rendered malesterile to prevent self pollination. This can be achieved genetically,manually or chemically, as is described herein. It is preferable thatthe agronomically elite parent has a similar maturity date to thevariety HOI002 to ensure coincidence of the availability of pollen andreceptivity thereto. The resulting kernels can then be harvested fromthe plants.

B. F₁ Hybrid Corn Plant and Seed Production

[0283] Any time the inbred corn plant HOI002 is crossed with another,different, corn inbred, a first generation (F₁) corn hybrid plant isproduced. As such, an F₁ hybrid corn plant may be produced by crossingHOI002 with any second inbred maize plant. Therefore, any F₁ hybrid cornplant or corn seed that is produced with HOI002 as a parent is part ofthe present invention.

[0284] The goal of the process of producing an F₁ hybrid is tomanipulate the genetic complement of corn to generate new combinationsof genes that interact to yield new or improved traits (phenotypiccharacteristics). A process of producing an F₁ hybrid typically beginswith the production of one or more inbred plants. Those plants areproduced by repeated crossing of ancestrally related corn plants to tryto combine certain genes within the inbred plants.

[0285] Corn has a diploid phase, which means two conditions of a gene(two alleles) occupy each locus (position on a chromosome). If thealleles are the same at a locus, there is said to be homozygosity. Ifthey are different, there is said to be heterozygosity. In a completelyinbred plant, all loci are homozygous. Because many loci when homozygousare deleterious to the plant, in particular leading to reduced vigor,less kernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

[0286] Inbreeding requires sophisticated manipulation by human breeders.Even in the extremely unlikely event inbreeding rather thancrossbreeding occurred in natural corn, achievement of completeinbreeding cannot be expected in nature due to well known deleteriouseffects of homozygosity and the large number of generations the plantwould have to breed in isolation. The reason for the breeder to createinbred plants is to have a known reservoir of genes whose gametictransmission is predictable. The development of inbred plants generallyrequires at least about 5 to 7 generations of selfing. Inbred plants arethen cross-bred in an attempt to develop improved F₁ hybrids. Hybridsare then screened and evaluated in small scale field trials. Typically,about 10 to 15 phenotypic traits, selected for their potentialcommercial value, are measured.

[0287] When the inbred corn plant HOI002 is crossed with another inbredplant to yield a hybrid, the original inbred can serve as either thematernal or paternal plant. For many crosses, the outcome is the sameregardless of the assigned sex of the parental plants. However, there isoften one of the parental plants that is preferred as the maternal plantbecause of increased seed yield and production characteristics. Someplants produce tighter ear husks leading to more loss, for example dueto rot. There can be delays in silk formation that deleteriously affecttiming of the reproductive cycle for a pair of parental inbreds. Seedcoat characteristics can be preferable in one plant. Pollen can be shedbetter by one plant. Other variables can also affect preferred sexualassignment of a particular cross. In the case of the instant inbred,HOI002 can be crossed as either the male or female parent.

C. F₁ Hybrid Comparisons

[0288] As mentioned above, hybrids are progressively eliminatedfollowing detailed evaluations of their phenotype, including formalcomparisons with other commercially successful hybrids. Strip trials areused to compare the phenotypes of hybrids grown in as many environmentsas possible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

[0289] In one aspect of the invention, comparative data may be used toassess hybrids produced using HOI002 as one parent. In one embodiment ofthe invention, the data may comprise grain quality trait measurements,for example, analysis of percent endosperm by dry weight, percent embryoby dry weight, percent oil in the endosperm by dry weight, oil in theembryo by dry weight percent and oil in the kernel by dry weight.Alternatively, a per kernel analysis could be used, for example, bycomparing traits such as endosperm size, embryo size, endosperm+embryoweight, oil weight in endosperm, oil weight in the embryo and oil weightin the endosperm+embryo. Examples of hybrid comparisons carried out onHOI002 are presented below, in Tables 2-4. TABLE 2 Enhancement of oilcontent in three-way hybrids with HOI002 Grain Stalk Root Oil YieldMoisture Lodging Lodging Content Pedigree (Bu/A) (%) (%) (%) (%) LH310 ×RQ0084 172.5 20.5 4.6 4.5 5.8 (LH310 × HOI002) × 131.1 20.1 7.0 16.7 9.8RQ0084 LH310 × RQ0085 177.7 25.5 2.8 5.4 6.1 (LH310 × HOI001) × 156.523.7 7.3 4.5 8.9 RQ0085

[0290] TABLE 3 Comparison of conventional hybrids to a hybrid of HOI002Grain Stalk Root Oil Yield Moisture Lodging Lodging Content Pedigree(Bu/A) (%) (%) (%) (%) (LH227 × LH228) × 175.5 18.0 21.2 11.8 4.5 LH82(LH227 × LH228) × 172.7 17.1 14.8 12.8 4.0 LH172 (LH227 × LH228) × 192.516.8 6.0 7.0 3.9 LH176 (L11227 × LH228) × 173.7 17.8 9.3 17.0 9.8 HOI002

[0291] TABLE 3 Comparison of conventional hybrids to a hybrid of HOI002Grain Stalk Root Oil Yield Moisture Lodging Lodging Content Pedigree(Bu/A) (%) (%) (%) (%) (HC33 × LH198) × 127.5 18.1 13.5 0.0 4.2 LH59(HC33 × LHI98) × 149.5 19.1 6.1 2.4 4.3 LH185 (HC33 × LH198) × 143.219.9 13.0 3.5 4.7 LH283 (HC33 × LH198) × 128.2 20.3 11.2 13.4 9.9 HOI002

X. GENETIC COMPLEMENTS

[0292] The present invention provides a genetic complement of the inbredcorn plant variety designated HOI002. Further provided by the inventionis a hybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from HOI002 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

[0293] As used herein, the phrase “genetic complement” means anaggregate of nucleotide sequences, the expression of which defines thephenotype of a corn plant or a cell or tissue of that plant. By way ofexample, a corn plant is genotyped to determine a representative sampleof the inherited markers it possesses. Markers are alleles at a singlelocus. They are preferably inherited in codominant fashion so that thepresence of both alleles at a diploid locus is readily detectable, andthey are free of environmental variation, i.e., their heritability is 1.This genotyping is preferably performed on at least one generation ofthe descendant plant for which the numerical value of the quantitativetrait or traits of interest are also determined. The array of singlelocus genotypes is expressed as a profile of marker alleles, two at eachlocus. The marker allelic composition of each locus can be eitherhomozygous or heterozygous. Homozygosity is a condition where bothalleles at a locus are characterized by the same nucleotide sequence orsize of a repeated sequence. Heterozygosity refers to differentconditions of the gene at a locus. A preferred type of genetic markerfor use with the invention is simple sequence repeats (SSRs), althoughpotentially any other type of genetic marker could be used, for example,restriction fragment length polymorphisms (RFLPs), amplified fragmentlength polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs),and isozymes.

[0294] A genetic marker profile of an inbred may be predictive of theagronomic traits of a hybrid produced using that inbred. For example, ifan inbred of known genetic marker profile and phenotype is crossed witha second inbred of known genetic marker profile and phenotype it ispossible to predict the phenotype of the F₁ hybrid based on the combinedgenetic marker profiles of the parent inbreds. Methods for prediction ofhybrid performance from genetic marker data is disclosed in U.S. Pat.No. 5,492,547, the disclosure of which is specifically incorporatedherein by reference in its entirety. Such predictions may be made usingany suitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

[0295] SSRs are genetic markers based on polymorphisms in repeatednucleotide sequences, such as microsatellites. A marker system based onSSRs can be highly informative in linkage analysis relative to othermarker systems in that multiple alleles may be present. Anotheradvantage of this type of marker is that, through use of flankingprimers, detection of SSRs can be achieved, for example, by thepolymerase chain reaction (PCR™), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR™ detection is done byuse of two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology. Following amplification,markers can be scored by gel electrophoresis of the amplificationproducts. Scoring of marker genotype is based on the size (number ofbase pairs) of the amplified segment.

[0296] Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. Thetechniques and nomenclature for isozyme analysis are described in, forexample, Stuber et al. (1988), which is incorporated by reference.

[0297] A standard set of loci can be used as a reference set.Comparative analysis of these loci is used to compare the purity ofhybrid seeds, to assess the increased variability in hybrids compared toinbreds, and to determine the identity of seeds, plants, and plantparts. In this respect, an isozyme reference set can be used to developgenotypic “fingerprints.”

[0298] The present invention also provides a hybrid genetic complementformed by the combination of a haploid genetic complement of the cornplant HOI002 with a haploid genetic complement of a second corn plant.Means for combining a haploid genetic complement from the foregoinginbred with another haploid genetic complement can comprise any methodfor producing a hybrid plant from HOI002. It is contemplated that such ahybrid genetic complement can be prepared using in vitro regeneration ofa tissue culture of a hybrid plant of this invention.

[0299] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of the foregoing illustrative embodiments,it will be apparent to those of skill in the art that variations,changes, modifications, and alterations may be applied to thecomposition, methods, and in the steps or in the sequence of steps ofthe methods described herein, without departing from the true concept,spirit, and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

[0300] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0301] Anderson, W. P., Weed Science Principles, West PublishingCompany, 1983.

[0302] Armstrong and Green, “Establishment and maintenance of friable,embryogenic maize callus and the involvement of L-proline,” Planta,164:207-214, 1985.

[0303] Conger, Novak, Afza, Erdelsky, “Somatic Embryogenesis fromCultured Leaf Segments of Zea Mays,” Plant Cell Reports, 6:345-347,1987.

[0304] Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,”Planta, 165:322-332, 1985.

[0305] Fehr, “Theory and Technique,” In: Principles of CultivarDevelopment, 1:360-376, 1987.

[0306] Gaillard et al., “Optimization of Maize Microspore Isolation andCulture Condition for Reliable Plant Regeneration,” Plant Cell Reports,10(2):55, 1991.

[0307] Gordon-Kamm et al., “Transformation of Maize Cells andRegeneration of Fertile Transgenic Plants,” The Plant Cell, 2:603-618,1990.

[0308] Green and Rhodes, “Plant Regeneration in Tissue Cultures ofMaize: Callus Formation from Stem Protoplasts of Corn (Zea Mays L.),”In: Maize for Biological Research, 367-372, 1982.

[0309] Jensen, “Chromosome Doubling Techniques in Haploids,” Haploidsand Higher Plants—Advances and Potentials, Proceedings of the FirstInternational Symposium, 1974.

[0310] Nienhuis et al., “Restriction Fragment Length PolymorphismAnalysis of Loci Associated with Insect Resistance in Tomato,” CropScience, 27(4):797-803, 1987.

[0311] Omirulleh et al., “Activity of a chimeric promoter with thedoubled CaMV 35S enhancer element in protoplast-derived cells andtransgenic plants in maize,” Plant Mol. Biol., 21(3):415-428, 1993.

[0312] Pace et al, “Anther Culture of Maize and the Visualization ofEmbryogenic Microspores by Fluorescent Microscopy,” Theoretical andApplied Genetics, 73:863-869, 1987.

[0313] Poehlman et al., “Breeding Field Crops,” 4th Ed., Iowa StateUniversity Press, Ames, Iowa, pp 132-155 and 321-344, 1995.

[0314] Rao et al., “Somatic Embryogenesis in Glume Callus Cultures,”Maize Genetics Cooperation Newsletter, 60, 1986.

[0315] Songstad et al., “Effect of 1-Aminocyclopropate-1-CarboxylicAcid, Silver Nitrate, and Norbornadiene on Plant Regeneration from MaizeCallus Cultures,” Plant Cell Reports, 7:262-265, 1988.

[0316] Sprague and Dudley (eds.), “Corn and Corn Improvement,” 3rd Ed.,Crop Science of America, Inc., and Soil Science of America, Inc.,Madison Wis. pp 881-883 and pp 901-918, 1988.

[0317] Stuber et al., “Techniques and scoring procedures for starch gelelectrophoresis of enzymes of maize C. Zea mays, L.,” Tech. Bull., 286,1988.

[0318] Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus,”Theoretical and Applied Genetics, 77:889-892, 1989.

[0319] Wang et al., “Large-Scale Identification, Mapping, and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082, 1998.

[0320] Williams et al., “Oligonucleotide Primers of Arbitrary SequenceAmplify DNA Polymorphisms which Are Useful as Genetic Markers,” NucleicAcids Res., 18:6531-6535, 1990.

What is claimed is:
 1. A seed of the corn variety HOI002, wherein asample of the seed of the corn variety HOI002 was deposited under ATCCAccession No. PTA-3788.
 2. A population of seed of the corn varietyHOI002, wherein a sample of the seed of the corn variety HOI002 wasdeposited under ATCC Accession No. PTA-3788.
 3. The population of seedof claim 2, further defined as an essentially homogeneous population ofseed.
 4. The population of seed of claim 2, further defined asessentially free from hybrid seed.
 5. A corn plant produced by growing aseed of the corn variety HOI002, wherein a sample of the seed of thecorn variety HOI002 was deposited under ATCC Accession No. PTA-3788. 6.A plant part of the corn plant of claim
 5. 7. The plant part of claim 6,further defined as pollen.
 8. The plant part of claim 6, further definedas an ovule.
 9. The plant part of claim 6, further defined as a cell.10. A tissue culture comprising the cell of claim
 9. 11. An essentiallyhomogeneous population of corn plants produced by growing the seed ofthe corn variety HOI002, wherein a sample of the seed of the cornvariety HOI002 was deposited under ATCC Accession No. PTA-3788.
 12. Acorn plant capable of expressing all the physiological and morphologicalcharacteristics of the corn variety HOI002, wherein a sample of the seedof the corn variety HOI002 was deposited under ATCC Accession No.PTA-3788.
 13. The corn plant of claim 12, further comprising a nuclearor cytoplasmic gene conferring male sterility.
 14. A tissue culture ofregenerable cells of a plant of corn variety HOI002, wherein the tissueis capable of regenerating plants capable of expressing all thephysiological and morphological characteristics of the corn varietyHOI002, wherein a sample of the seed of the corn variety HOI002 wasdeposited under ATCC Accession No. PTA-3788.
 15. The tissue culture ofclaim 14, wherein the regenerable cells comprise cells derived fromembryos, immature embryos, meristematic cells, immature tassels,microspores, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.
 16. The tissue culture of claim15, wherein the regenerable cells comprise protoplasts or callus cells.17. A corn plant regenerated from the tissue culture of claim 14,wherein the corn plant is capable of expressing all of the physiologicaland morphological characteristics of the corn variety designated HOI002,wherein a sample of the seed of the corn variety HOI002 was depositedunder ATCC Accession No. PTA-3788.
 18. A process of crossing cornplants, comprising crossing a first parent corn plant with a secondparent corn plant, wherein one or both of the first or the second parentcorn plant is a plant of the corn variety HOI002, wherein a sample ofthe seed of the corn variety HOI002 was deposited under ATCC AccessionNo. PTA-3788, wherein kernels are allowed to form.
 19. The process ofclaim 18, wherein the kernels are used as grain.
 20. A process ofproducing corn grain, comprising: (a) planting a population of cornplants comprising first and second corn plants in pollinating proximity;wherein the first plant is an agronomically elite variety and whereinthe second plant is the corn plant variety HOI002, wherein a sample ofthe seed of the corn variety HOI002 was deposited under ATCC AccessionNo. PTA-3788; (b) growing the first and second plants to maturity; (c)allowing pollen from the second plant to pollinate the first plant; and(d) collecting seed that forms on at least the first plant.
 21. Theprocess of claim 20, wherein collecting comprises harvesting seed formedon the first corn plant and the second corn plant.
 22. The process ofclaim 20, wherein the first corn plant is genetically male sterile. 23.The process of claim 20, wherein planting comprises randomlyinterplanting the first corn plant and the second corn plant within saidpopulation.
 24. A process of producing hybrid corn seed, comprisingcrossing a first inbred corn plant with a second, distinct inbred cornplant, wherein the first or second inbred corn plant is a plant of thecorn variety HOI002, wherein a sample of the seed of the corn varietyHOI002 was deposited under ATCC Accession No. PTA-3788.
 25. The processof claim 24, wherein crossing comprises the steps of: (a) planting theseeds of first and second inbred corn plants; (b) cultivating the seedsof said first and second inbred corn plants into plants that bearflowers; (c) preventing self pollination of at least one of the first orsecond inbred corn plant; (d) allowing cross-pollination to occurbetween the first and second inbred corn plants; and (e) harvestingseeds on at least one of the first or second inbred corn plants, saidseeds resulting from said cross-pollination.
 26. A hybrid corn seedproduced by the process of claim
 25. 27. A hybrid corn plant produced bygrowing a seed produced by the process of claim
 25. 28. The hybrid cornplant of claim 27, wherein the plant is a first generation (F₁) hybridcorn plant.
 29. The corn plant of claim 5, further defined as having agenome comprising a single locus conversion.
 30. The corn plant of claim29, wherein the single locus was stably inserted into a corn genome bytransformation.
 31. The corn plant of claim 29, wherein the locus isselected from the group consisting of a dominant allele and a recessiveallele.
 32. The corn plant of claim 29, wherein the locus confers atrait selected from the group consisting of herbicide tolerance, insectresistance, resistance to bacterial, fungal, nematode or viral disease,yield enhancement, waxy starch, improved nutritional quality, enhancedyield stability, male sterility and restoration of male fertility.
 33. Aprocess of producing a corn plant derived from the corn plant of claim5, the process comprising the steps of: (a) preparing a progeny plantderived from corn variety HOI002 by crossing a plant of the corn varietyHOI002 with a second corn plant, wherein a sample of the seed of thecorn variety HOI002 was deposited under ATCC Accession No. PTA-3788; and(b) crossing the progeny plant with itself or a second plant to producea progeny plant of a subsequent generation.
 34. The process of claim 33,further defined as a process of producing an inbred corn plant derivedfrom the corn plant of claim 5, comprising: (c) crossing the progenyplant of a subsequent generation with itself or a second plant; and (d)repeating steps (b) and (c) for an additional 2-10 generations toproduce an inbred corn plant derived from the corn variety HOI002.